Type 1 diabetes (formerly referred to as juvenile-onset or insulin-dependent diabetes) is an autoimmune disease resulting from destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. Insulin injections allow for continuation of life for the one million Americans afflicted with type 1 diabetes; however, they neither cure the disease nor prevent its devastating complications of microangiopathy, nephropathy, and neuropathy that diminish quality of life and shorten the life expectancy of a person with diabetes by an average of 15 years. The nonobese diabetic (NOD) mouse provides a relevant model system for type 1 diabetes that shares many of the characteristics of the human disease. Importantly, patients and NOD mice both develop lymphocytic infiltration of pancreatic islets (insulitis) and subsequent beta cell destruction mediated by T lymphocytes. Studies utilizing the NOD mouse have shown that beta cell-autoreactive CD8 + T cells restricted to class I major histocompatibility complex (MHC) molecules are absolutely required for disease development. Such effectors are also suspected contributors to beta cell elimination in both new-onset and graft-recurrent type 1 diabetes patients. Despite their importance, very little is known regarding the antigenic specificities of the CD8+ T cells participating in the initial phase of the a cell destruction that ultimately leads to disease. The overall goal of this proposal is to define these initiating specificities for T cells restricted to either murine or human class I MHC molecules. CD8 + T cells isolated from early insulitic lesions of either standard NOD mice or NOD mice transgenically expressing HLA-A2 will be utilized for this purpose. A combination of biochemical and expression cloning methods will be employed to identify the peptides recognized by these T cells and the proteins from which they are derived. Three Specific Aims are proposed: (1) To biochemically isolate and sequence the class I MHC-bound a cell peptides recognized by CD8 + T cell clones derived from early insulitic lesions of standard NOD mice; (2) To directly identify the autoantigens targeted by early insulitic CD8 + T cells in standard NOD mice using an expression cloning approach to complement the peptide isolation studies proposed in Aim 1; (3) To use NOD.HLA-A2.1 mice to identify the antigens recognized in the context of the human class I MHC molecule HLA-A2 during the early prediabetic period. Completion of the proposed Aims will result in an improved understanding of the pathogenesis and molecular basis of type 1 diabetes and could conceivably suggest new antigen-based intervention strategies and assays to monitor the autoimmune status of patients.